The goal of this research is to determine the molecular bases of congenital antithrombin III (ATIII) deficiency. Antithrombin III is an endogenous anticoagulant protein which plays a major role in maintaining the fluidity of blood. Genetic and acquired deficiencies of ATIII predispose the affected individual to thrombosis. The cDNA for human antithrombin III has been recently cloned (Bock et al., submitted) and will allow us to systematically and directly investigate mutations causing hereditary thrombosis, using the powerful techniques of classical genetic and molecular genetic analysis. A variety of ATIII mutations will be surveyed by whole genome Southern analysis of DNAs from affected individuals in many different deficiency pedigrees. This experiment will reveal mutant genes which are associated with deletions and DNA rearrangements, as well as mutations which appear to be "grossly normal". Mutations in the latter category can be distinguished as linked or unlinked to the ATIII structural locus by experiments in which segregation of ATIII-associated restriction fragment length polymorphisms (RFLPS) is followed in deficiency families. RFLP haplotype information generated by such studies will also be useful for predicting whether mutant genes of the many deficiency pedigrees represent the same or different DNA lesions. On the basis of criteria discussed above, specific ATIII mutations will be selected for detailed examination and isolated by molecular cloning. These genes will be characterized by mapping, in vitro functional assays, and nucleotide sequencing as appropriate, so as to determine the levels of biosynthesis at which antithrombin gene expression is aberrant and reveal the primary gene defects. Finally, these ATIII gene characterization and RFLP linkage studies will provide a foundation for development of prenatal diagnostic tests for hereditary thrombosis.